High throughput knockout constructs in yeast by homologous recombination

ABSTRACT

The present invention is directed to methods for producing gene targeting constructs by homologous recombination using mouse genomic libraries arrayed in shuttle vectors. The invention is also directed to methods of using targeting constructs made by the methods to generate transgenic animals.

FIELD OF THE INVENTION

[0001] The present invention relates to generation of genomic librariesin shuttle vectors and high throughput generation of vectors byhomologous recombination for creating transgenic animals by homologousrecombination.

BACKGROUND OF THE INVENTION

[0002] A particularly productive approach to understanding the functionof a particular gene in animals involves the disruption of the gene'sfunction by “targeted mutagenesis”. A common form of targetedmutagenesis is to generate “gene knockouts”. Typically, a gene knockoutinvolves disrupting a gene in the germline of an animal at an earlyembryonic stage. (See, Thomas et al., Cell, 51:503 (1987).) Onceestablished in the germline, it is possible to determine the effect ofthe mutation on the animal in both the heterozygous and homozygousstates by appropriate breeding of mice having the germline mutation.

[0003] The mouse knockout model system is very useful for functionalgenomic analysis of genes. The advantages of mouse models for the studyof mammalian physiology, and testing of therapies for the treatment ofhuman diseases, and developmental abnormalities have been extensivelyestablished.

[0004] Among the many examples of the use of knockout technologyutilized to investigate gene function are U.S. Pat. Nos. 5,625,122 and5,530,178 to Mak, T. which describe the production of mice having adisrupted gene encoding lymphocyte-specific tyrosine kinase p56^(lck)and Lyt-2, respectively. Silva et al., Science, 257:201 (1992) producedmice having a disrupted α-Calcium Calmodulin kinase II gene (αCaMKIIgene) which resulted in animals having an abnormal fear response andaggressive behavior. (See, also, Chen et al., Science, 266:291 [1994]).Wang et al., Science, 269:1108 (1995) demonstrated that the disruptionin mice of the C/EPBα gene which encodes a basic leucine zippertranscription factor results in impaired energy homeostasis in themutant animals. Knudsen et al., Science, 270:960 (1995) demonstratedthat disruption of the BAX gene in mice results in lymphoid hyperplasiaand male germ cell death.

[0005] The most common approach to producing knockout animals involvesthe disruption of a target gene by inserting into the target gene(usually in embryonic stem cells), via homologous recombination, a DNAconstruct encoding a selectable marker gene flanked by DNA sequenceshomologous to part of the target gene. When properly designed, the DNAconstruct effectively integrates into and disrupts the targeted genethereby preventing expression of an active gene product encoded by thatgene.

[0006] Homologous recombination involves recombination between twogenetic elements (either extrachromosomally, intrachromosomally, orbetween an extrachromosomal element and a chromosomal locus) viahomologous DNA sequences, which results in the physical exchange of DNAbetween the genetic elements. Homologous recombination is not limited tomammalian cells but also occurs in bacterial cells, yeast cells, in theslime mold Dictyostelium discoideum and in other organisms. For a reviewof homologous recombination in mammalian cells, see Bollag et al., Ann.Rev. Genet., 23:199-225 (1989) (incorporated herein by reference). For areview of homologous recombination in fungal cells, see Orr-Weaver etal., Microbiol. Reviews, 49:33-58 (1985) incorporated herein byreference.

[0007] With the increasing awareness that animal, and particularly mousemutations can provide such useful insights about the function of genesfrom humans, a great deal of interest is developing to systematicallygenerate mutations within genes in mice that correspond to those geneswhich are being isolated and characterized as part of various genomeinitiatives such as the Human Genome Project. The problem with utilizingthese procedures for large-scale mutagenesis experiments is that thetechnologies for generating transgenic animals and targeted mutationsare currently very tedious, expensive, and labor intensive. The mosttedious parts of making an animal knockout construct from a given cDNAis obtaining an appropriate genomic fragment and gene mapping. Once thegenomic fragment is obtained and mapped, actual assembly of thetargeting vector also is a tedious process depending upon availabilityof appropriate restriction sites.

[0008] Generally, the preparation of these constructs requires isolatinggenomic clones containing the region of interest, developing restrictionmaps, engineering restriction sites into the clones, and restrictiondigesting and ligating fragments to engineer the specific constructneeded to produce the knockout. See, e.g., Mak, T. U.S. Pat. Nos.5,625,122 and 5,530,178; Joyner et al., Nature, 338:153-156 (1989);Thomas et al., supra; Silva et al., supra, Chen et al., supra; Wang etal., supra; and Knudsen et al., supra. This is a long and tediousprocess that can take several months to complete. Thus, in order to morerapidly and efficiently create model organisms with genomicmodifications, there exists a need to develop high throughput methodsfor the production of targeting constructs which do not requireidentification of target genomic fragments by traditional means, theircloning, and subsequent restriction mapping and other complex molecularengineering steps.

SUMMARY OF THE INVENTION

[0009] The present invention, in preferred embodiments, provides methodsof preparing a genomic library for use in producing knockout targetingvectors comprising preparing a size selected mouse genomic DNA;preparing a shuttle vector comprising inserting said genomic DNA into ayeast vector, wherein the vector comprises a first bacterial origin ofreplication; a first bacterial selection marker; a first yeast origin ofreplication; a first yeast selection marker; and a first mammalianselection marker; transforming bacterial host cells with said shuttlevector to amplify said genomic library; arraying said transformed hostcells into pools of cloned cells comprising shuttle vectors comprising agenomic DNA fragment; a first yeast origin of replication; a first yeastselection marker; a first bacterial origin of replication; a firstbacterial selection marker; and a first selection marker for integrationinto mammalian cells; wherein the cells in said pools comprise mousegenomic fragments of different size.

[0010] In specific embodiments, the genomic DNA is a library whichcomprises mouse genomic DNA fragments ranging from about 8 kb to about14 kb. More particularly, the mouse genomic DNA fragments are isolatedfrom a mouse strain selected from the group consisting of 129svj, 129Ola, 129sv, and C57BL/6. Of course, these are merely exemplary strainsof mice and those of skill in the art will be aware that other mousestrains may be employed for generating the transgenic animals of thepresent invention. Likewise, while certain preferred embodiments aredirected to the generation of transgenic mice, it should be understoodthat the present invention is equally applicable to generatingtransgenic animals of other species such as, for example, mammalsincluding but not limited to rabbits, mice, rats, hamsters, goats,sheep, pigs, horses, cows, dogs, cats, as well as primates, such as,monkeys, apes, and baboons.

[0011] In specific embodiments, the genomic library, when transformedinto the bacterial host cells with said shuttle vector generates betweenabout 3×10⁶ and 5×10⁶ clones. This is an exemplary range and it iscontemplated that those of skill in the art may prepare a genomiclibrary that generates more or fewer clones. Thus the practice of theinvention may generate about 1×10⁶ clones, about 2×10⁶ clones, about3×10⁶ clones, about 4×10⁶ clones, about 5×10⁶ clones, about 6×10⁶clones, about 7×10⁶ clones, about 8×10⁶ clones, about 9×10⁶ clones,about 10×10⁶ clones or more clones or indeed may generate less than1×10⁶ clones and still provide meaningful shuttle vectors that may beused in the context of the present invention. The host cells that areavailable for transformation can be any host cell well known to those ofskill in the art. In preferred embodiments, the host cells are bacterialcells selected from the group consisting of Escherichia coli, Bacillussubtilis, Pseudomonas aeruginosa, Salmonella typhimurium and Serratiamarcescans. Particularly preferred host cells are E. coli.

[0012] In preferred embodiments, the shuttle vector comprises abacterial origin of replication selected from the group consisting ofColE1-ORI, F and R1. Preferably, the bacterial origin of replication isan E. coli origin of replication. In specific embodiments, the E. coliorigin of replication is ColE1-ORI. The yeast origin of replicationpreferred in the context of the preferred vectors of the presentinvention is selected from the group consisting of Cen, 2 m and theautonomous replication sequence.

[0013] The shuttle vectors may employ any selectable marker commonlyused to monitor bacterial propagation, yeast propagation and selectionin mammalian cells. In preferred embodiments, the marker for bacterialpropagation is selected from the group consisting of ampicillinresistance, tetracycline resistance, neomycin resistance, kanamycinresistance and chloramphenicol resistance. These are merely exemplaryand additional markers will be well known to those of skill in the artand are contemplated to be useful in the present invention. In preferredembodiments, the bacterial propagation marker for ampicillin resistanceis BlaI.

[0014] Those of skill in the art will understand that any yeastselectable marker may be advantageously employed in the shuttle vectorsof the present invention. In preferred embodiments, the marker forpropagation in yeast is selected from the group consisting of trpl, His,Ura3, Arg, Ade and Leu2. The selectable marker for mammalian cells maybe selected from the group consisting of neomycin resistance, hygromycinresistance, zeocin resistance, Salmonella HisD and puromycin N-acetyltransferase. In certain embodiments, the vectors may further comprise anegative selectable marker. In specific embodiments, the negativeselectable marker is selected from the group consisting of thymidinekinase, and xanthine-guanine-phosphoribosyltransferase.

[0015] In particularly preferred embodiments, the yeast vector of thepresent invention comprises a BamHI site for inserting said genomicfragments. More particular embodiments contemplate that the BamHI siteis flanked by priming sequences to facilitate PCR amplification.Generally, priming sequences for PCR amplification are well known tothose of skill in the art; preferably, the priming sequences are Sp6 andT7 priming sequences. In particularly preferred embodiments, the yeastvector of the present invention is the vector designated as pYYL-1.

[0016] In specific aspects the shuttle vector further comprises rarecutting enzyme sites flanking the genomic fragment. More specifically,the shuttle vector comprises rare cutting enzyme sites flanking themammalian selection marker. In preferred aspects of the presentinvention, the mouse genomic library described by the present inventionis used for high throughput construction of knockout vectors, and morespecifically mouse knockout vectors.

[0017] Another aspect of the present invention contemplates a method forthe preparation of a gene targeting vector for homologous recombinationcomprising selecting a bacterial clone pool positive for the gene to betargeted from an array of bacterial clones comprising the mouse genomiclibrary described above; isolating the DNA from said positive pool;preparing a second expression construct comprising a marker cassettecomprising a second yeast selectable marker and a second mammalianselectable marker, wherein said marker cassette is flanked on each sideby mammalian gene-specific sequences homologous for a portion of thegene to be targeted; transforming yeast cells with the second expressionconstruct and the DNA from the positive clone; selecting the transformedyeast cells for expression of the first and second yeast selectablemarkers; and isolating the targeting vector produced by therecombination between the shuttle vector and the second expressionconstruct.

[0018] In specific embodiments, the positive pools comprising the targetgene are selected by PCR analysis of the pools with gene-specific PCRprimers wherein amplification of the PCR products is indicative of thepool comprising the target gene of interest. PCR amplificationtechniques are well known to those of skill in the art and are describedin for example Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0019] In preferred embodiments, the gene-specific flanking sequenceseach comprises at least about 20 nucleotides. In other embodiments , thegene specific flanking sequences each comprises from about 35 to about400 nucleotides. This is an exemplary range and it is contemplated thatthe gene specific flanking sequences may comprise for example, about 20,about 25, about 30, about 35, about 40, about 45, about 50, about 55,about 60, about 65, about 70, about 75, about 80, about 85, about 90,about 95, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170, about 180, about 190, about 200, about 210,about 220, about 230, about 240, about 250, about 260, about 270, about280, about 290, about 300, about 310, about 320, about 330, about 340,about 350, about 360, about 370, about 380, about 390, about 400, about450, about 500, about 550, about 600, about 650, about 700, about 750,about 800, about 850, about 900, about 950, about 1000 or morenucleotides.

[0020] In specific embodiments, the fragment of genomic DNA comprisesfrom about 0.5 kb to about 5 kb of DNA on each side of a site in saidgene to be targeted. This is merely an exemplary range and it iscontemplated that the fragment of genomic DNA may comprise about 0.5 kb,0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1.0 kb, 1.2 kb, 1.4 kb, 1.5 kb, 1.6 kb,1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.25 kb, 2.5 kb, 2.75 kb, 3.0 kb, 3.25kb, 3.5 kb, 3.75 kb, 4.0 kb, 4.25 kb, 4.5 kb, 4.75 kb, 5.0 kb, 5.5 kb,6.0 kb, 6.5 kb, 7.0 kb or more of DNA on each side of a site in saidgene to be targeted. In particularly preferred embodiments, the fragmentof genomic DNA comprises at least about 1 kb of genomic DNA on each sideof a site in said gene to be targeted. In certain embodiments, thesecond yeast selectable marker is selected from the group consisting ofTRP1, His, Ura3, Ade, Arg and Leu2. In other embodiments, the secondmammalian selectable marker is selected from the group consisting ofconsisting of thymidine kinase, neomycin resistance, hygromycinresistance, Salmonella HisD and puromycin N-acetyl transferase. Inspecific embodiments, it is contemplated that the marker cassettecomprises Ura3 as the second yeast selectable marker and the neomycinresistance gene as the second mammalian selectable marker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of this application and areincluded to further demonstrate certain aspects of the invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of the specificembodiments of the present invention.

[0022]FIG. 1 shows a flow diagram describing the construction andarraying of mouse genomic library into a yeast-E. Coli shuttle vector.

[0023]FIG. 2 shows a flow diagram describing the construction ofknock-out vectors from pooled mouse genomic library by using yeasthomologous recombination.

[0024]FIG. 3 shows a flow diagram describing generation of a recombinedknockout cassette according to the present invention.

[0025]FIG. 4 shows a map of the pYYL-1 mouse knock-out vector.

[0026]FIG. 5 shows a map of a targeting vector for a GPR24 knock-out.

[0027]FIG. 6 shows a map of a targeting vector for a CHL-1 knock-out.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Traditional methods of generating knockout animals involve theisolation of a specific genomic sequence from a given animal forexample, a mouse which is then employed in the specific targetingconstruct to generate the appropriate knockout vector. In suchconventional method, cDNA is used to obtain a corresponding genomicbacterial artificial chromosome (BAC) clone/s, followed by extensivemapping and Southerns for selection of appropriate-sized fragments.These fragments must then be sub-cloned into an appropriate vector forconstruction of knockout cassette. Appropriate restriction enzyme sitesmust be present in order to remove the targeted area within the codingregion and to replace it with a mammalian selection marker. In addition,this process of identifying BACs, subcloning and making conventionalknockout cassette must be repeated for each knockout desired. Thistraditional technique is laborious and time consuming.

[0029] The present invention reduces the number of cloning stepsrequired for creating a mouse knock-out targeting vector to a singleinsertion of a selection cassette by using a mouse genomic library and arecombination cassette. Prior knowledge of genomic structure of the geneof interest is not required, which is another major advantage of thesystem.

[0030] More particularly, in the present invention, homologousrecombination in yeast in a high throughput setting is employed toconstruct a mouse genomic library in yeast-E. coli shuttle vector(s).Construction of the mouse knock-out targeting vector is then achieved ina single homologous recombination step using the mouse genomic libraryin the yeast vector. This method is advantageous in that it provides areadily available library for the generation of knockout constructs,thereby saving time and cost.

[0031] The method described herein allows the generation of knockouttargeting vectors within two to three weeks beginning with the screeningof the arrayed genomic library in the E. coli-yeast shuttle-vector. Inpreferred aspects the arrayed genomic library is generated from a mouse,however, as stated above the techniques described herein will be usefulfor the production of other transgenic animals. Vectors generated bythis method allow both positive and negative selection. Positiveselection is applied to select for cells that stably integrate thepositive selection marker of the targeting vector. Negative selection isapplied to select against those cells in which either an integration ora non-homologous recombination event causes incorporation of thenegative selection cassette. Methods and compositions for preparing thelibrary, targeting constructs and transgenic animals are described infurther detail.

[0032] I. Homologous Recombination using Arrayed Genomic Libraries

[0033] The present invention is directed to providing more efficientmethods of creating vectors using homologous recombination. Moreparticularly the present invention discloses methods of making and usingmouse genomic libraries arrayed in shuttle vectors to create targetingvectors for producing knock-out mice.

[0034] Homologous recombination relies on the tendency of nucleic acidsto base pair with complementary sequences. In this instance, the basepairing serves to facilitate the interaction of two separate nucleicacid molecules so that strand breakage and repair can take place. Inother words, the “homologous” aspect of the method relies on sequencehomology to bring two complementary sequences into close proximity,while the “recombination” aspect provides for one complementary sequenceto replace the other by virtue of the breaking of certain bonds and theformation of others.

[0035] Put into practice, homologous recombination in the context of thepresent invention is used as follows. First, a target gene is selectedwithin the host cell. Sequences homologous to the target gene are thenincluded in a genetic construct, along with some mutation that willmodify the activity of the target gene (stop codon, interruption,deletion, constitutive mutation, etc.). The homologous sequences oneither side of the modifying mutation are said to “flank” the mutation.Flanking, in this context, simply means that target homologous sequencesare located both upstream (5′) and downstream (3′) of the mutation.These sequences should correspond to some sequences upstream anddownstream of the target gene. The construct is then introduced into thecell, thus permitting recombination between the cellular sequences andthe construct.

[0036] Targeted mutagenesis of a gene will result in an alteration(e.g., partial or complete inactivation or constitutivity) of normalproduction or structure of the polypeptide encoded by the targeted geneof a single cell, selected cells or all of the cells of an animal (or inculture) by introducing an appropriate targeting construct into a sitein the gene to be disrupted.

[0037] Targeted mutagenesis may also refer to “knocking in” a gene whichmeans replacing one gene with all or part of another gene from the sameor a heterologous organism for the purpose of determining, for example,whether two genes are functionally equivalent (see, e.g., Hanks et al.,Science, 269:679 (1995), incorporated herein by reference), althoughother applications are possible. For example, transcriptional regulatorysequences such as promoters may be knocked in to a region of a genome soas to be operatively linked to a structural sequence.

[0038] As a practical matter, the genetic construct will normally act asfar more than a vehicle to interrupt the gene. For example, it isimportant to be able to select for recombinants and, therefore, it iscommon to include within the construct a selectable marker gene. Thisgene permits selection of cells that have integrated the construct intotheir genomic DNA by conferring resistance to various biostatic andbiocidal drugs. In addition, a heterologous gene that is to be expressedin the cell also may advantageously be included within the construct.The arrangement may be as follows:

[0039] . . . vector . . . 5′-flanking sequence . . . heterologous gene .. . selectable marker gene . . . flanking sequence-3′ . . . vector . . .

[0040] Thus, using this kind of construct, it is possible, in a singlerecombinatorial event, to (i) “knock out” an endogenous gene, (ii)provide a selectable marker for identifying such an event and (iii)introduce a heterologous gene for expression.

[0041] In most cases, targeting constructs are constructed so as toinclude at least a portion of a gene to be disrupted. Typically, theportion of the gene included in the targeting construct is interruptedby insertion of a marker sequence (usually a selectable marker) thatdisrupts the reading frame of the interrupted gene so as to precludeexpression of an active gene product. This most often causes a knock outor inactivation of a gene.

[0042] Another refinement of the homologous recombination approachinvolves the use of a “negative” selectable marker. This marker, unlikethe selectable marker, causes death of cells which express the marker.Thus, it is used to identify undesirable recombination events. Whenseeking to select homologous recombinants using a selectable marker, itis difficult in the initial screening step to identify proper homologousrecombinants from recombinants generated from random, non-sequencespecific events. These recombinants also may contain the selectablemarker gene and may express the heterologous protein of interest, butwill, in all likelihood, not have the desired “knock out” phenotype. Byattaching a negative selectable marker to the construct, but outside ofthe flanking regions, one can select against many random recombinationevents that will incorporate the negative selectable marker. Homologousrecombination will likely not introduce the negative selectable marker,as it is outside of the flanking sequences.

[0043] Thus, for preparing knockouts, a gene within a host cell ischosen as the target gene into which a selection marker gene is to betransferred. Sequences homologous to the target gene are included in theexpression vector, and the selection gene is inserted into the vectorsuch that target gene homologous sequences are interrupted by theselection gene or, put another way, the target gene homologous sequences“flank” the selection gene. In preferred embodiments, a drug selectablemarker gene also is inserted into the target gene homologous sequences.Given this possibility, it should be apparent that the term “flank” isused broadly herein, namely, as describing target homologous sequencesthat are both upstream (5′) and downstream (3′) of the heterologous geneand/or the drug selectable marker gene. In effect, the flankingsequences need not directly abut the genes they “flank.” Application ofa drug to such cells will permit isolation of recombinants, in thatexpression of the marker in the cells will confer drug resistancewhereas cells that do not express that targeting sequence will not beresistant to the drug and will die when grown in the presence of thedrug.

[0044] Similarly, targeting constructs designed for knocking in genescan recombine at the homologous genomic site by homologous recombinationand will result in the introduction of all or a portion of a gene intothat locus. Techniques for knocking in genes are described in detail inHanks et aL., Science, 269:679 (1995) which is incorporated herein byreference. Methods for homologous recombination specific to the presentinvention and methods of introducing the knockout mutation into thegermline of an animal are described in further detail below.

[0045] Practice of the invention involves preparing genomic library froma selected mouse strain in yeast-E. coli shuttle vector and arecombination cassette for producing the targeting vector by homologousrecombination. The invention preferably involves preparing at least twoDNA constructs, a yeast shuttle vector comprising the genomic DNA from aselected mouse strain and a recombinant cassette for producing thehomologous recombination.

[0046] The yeast shuttle vector comprising the library of DNA fragmentsisolated from the mouse strain generally comprises a yeast selectablemarker, a bacterial selectable marker and a mouse genomic DNA fragment.The mouse genomic DNA is size fractionated into fragments of betweenabout 8 kb to about 14 kb of the total mouse genomic DNA. As such, thefragments may contain all or a portion of any number of genes.

[0047] The recombination-cassette of the present invention generallywill comprise unique flanking sequences which are different from oneanother and which correspond to sequences in the genomic site that is tobe targeted. Interposed between the flanking sequences may be positioneda sequence that encodes a marker. This sequence will thus have a twofold function in that it will act as a marker for the homologousrecombination as well as acting as a disruption sequence. Alternatively,a transcriptional regulatory sequence or a combination of a markersequence disposed 53 to a transcriptional regulatory sequence may beinterposed between the flanking sequences.

[0048] The genomic library in the yeast shuttle vector and therecombination cassette are introduced into yeast cells which mediaterecombination between the homologous sequences in the shuttle vector andthe recombination cassette, effectively introducing the DNA interposedbetween the unique flanking DNA into the fragment of genomic DNA in theshuttle vector. The resulting targeting construct may be used, asdescribed above, to produce targeted mutations.

[0049] It should be noted that the DNA sequences involved in homologousrecombination according to any aspect of the present invention need notbe 100% homologous with one another (or identical), however in general,the greater the homology between sequences the greater the efficiency ofrecombination.

[0050] In preferred aspects of the present invention, to generate therecombination cassette for any gene of interest, two (i.e., a sense andantisense) oligonucleotide primers are synthesized of which 45nucleotides are homologous to the targeted area of the gene of interest.In addition to the homology to the target region, the sense primer atits 3′ end also contains additional 20 bp that correspond to 5′ end ofthe neomycin gene. In the antisense primer, 20 bp at the 3′ end arehomologous to the 3′ end of the URA3 gene. Using these primers, a 2.8 kbcassette is generated that has 45 bp flanking sequences at its ends thatare homologous to the gene of interest. This homology is used fordirected recombination of the cassette with the gene of interest withinthe mouse genomic library in yeast.

[0051] The yeast Saccharomyces cerevisiae has highly developed geneticsystems involving homologous recombination that have been very usefulfor genetic engineering in vivo (see, e.g., Orr-Weaver et al, MicrobiolRev., 49:33 [1985]). In yeast, linear double-stranded (ds) DNA undergoesefficient homologous recombination with either chromosomal or plasmidtargets (Orr-Weaver et al., supra). The present invention is directed toexploiting the yeast homologous recombination system in order toincrease the efficiency of production of targeting constructs for thegeneration of targeted mutations (e.g., knock out or knock inmutations). Specifically, yeast-E. coli shuttle vectors are generated inwhich the whole mouse genomic DNA in fragments of between about 8 kb toabout 14 kb is arrayed in pools of E. coli clones.

[0052] According to the present invention, homologous recombination inyeast allows the preparation of targeting constructs to targetessentially any segment of the mouse or other mammalian genome. Unlikethe traditional methods used to make targeting constructs, the methodsof the present invention do not require detailed restriction mapping,convenient restriction sites, or the engineering of restriction sites,but instead use a genomic clone comprising a fragment containing atleast a part of an exon of a target gene or a portion of the locus to betargeted (including 53 or 33 untranslated sequences or intronsequences). Using polymerase chain reaction (PCR) primers to determinewhether a particular clone comprises such a targeted gene will allow oneof skill in the art to produce knock-out targeting vectors with limitedsequence information regarding the exon or locus of the target gene. Theapproach is exemplified below with reference to particular genes andparticular mouse strains, however, the methods of the present inventionare readily adaptable to other genes and other species of mice and othermammals. The general method of the invention is depicted schematicallyin FIGS. 1 through 3 in which FIG. 1 describes the construction andarraying of the mouse genomic library in pools of clones and FIGS. 2 and3 describe the use of the genomic library to create knock out vectorsfor homologous recombination.

[0053] By way of overview and with reference to FIGS. 1 through 3, alibrary of fragments of mouse 129/Svj (see, e.g., Knudsen et al.,Science, 270:96 (1995)) genomic DNA obtained from a genomic library of129/sv DNA containing at least part of the gene to be targeted is clonedinto a yeast/E. coli shuttle vector which has selectable markersallowing selection in yeast, E. coli and in mammalian cells. A selectionor marker gene generally encodes a polypeptide, which allows formaintenance of the plasmid in a population of cells. Some selectionmarkers can also be used negatively in which loss of the marker confersviability to the host cells under certain growth conditions. Typicalproteins include those that confer resistance to antibiotics or othertoxins or allow growth in the presence of specific nutrients.

[0054] Markers for selection in yeast are well known to those of skillin the art and include those involved in growth on specific sugar andamino acid substrates, such as trp, ura, leu, ade and his genes, whichprovide for maintenance of the plasmid in transformed yeast host cellslacking the corresponding functional genes on the host chromosome.Markers for selection in bacterial cells such as E. coli include thoseconferring resistance to antibiotics such as ampicillin,chloramphenicol, kanamycin, and the like. Positive and negative markersfor selection of mammalian cells also will be used. Positive markergenes functional in mammalian cells include neomycin resistance, zeocinresistance, and hygromycin resistance markers, whereas negativeselection markers may include TK (thymidine kinase) and XGPRT(xanthine-guanine-phosphoribosyltransferase).

[0055] Generally, negative selection markers may code for enzymes, whichconvert nucleotide analogs to products which are lethal uponincorporation into DNA. More particularly, TK is a versatile selectionmarker because cells can be selected for either loss or acquisition ofthis gene under different growth conditions. TK selection has provenuseful for generation of cellular and viral gene knockouts. The presenceof the thymidine kinase gene may be detected by the use of nucleosideanalogs such as acyclovir or Gancyclovir which will induce cytotoxiceffects on cells that contain a functional thymidine kinase gene. Theabsence of sensitivity to these nucleoside analogs indicates the absenceof the thymidine kinase gene.

[0056] In an exemplary embodiment, genomic DNA from the liver of 129svjmouse strain is isolated using the procedure described in MolecularCloning: A laboratory manual (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The mouse genomic DNA derived from other mouse strains such as 129 Ola,129sv, C57BL/6 also may be used for the purpose of making mouse genomiclibrary. The purified high-molecular-weight genomic DNA is partiallydigested with Sau3A restriction enzyme to randomly fragment the DNA andto obtain directly clonable ends. This DNA is then subjected to sizefractionation through a sucrose density gradient. (Maniatis , T. et al.(1978). The isolation of structural genes from libraries of eukaryoticDNA. Cell 15: 687). Additional methods of size fractionation also arecontemplated for the present invention. Such fractionation should yieldfractions containing 8-14 kb genomic fragments which are subsequentlycloned into the yeast-E. coli shuttle vector.

[0057] The shuttle vector into which the genomic DNA is cloned has to becapable of propagation in both yeast cells and in E. coli. In the yeastcells the shuttle vector will serve as a target for recombination andgeneration of the targeting vector and in the E. coli cells the shuttlevector will be amplified to obtain significant amounts of the shuttlevector. The shuttle vectors are prepared according to techniques wellknown to those of skill in the art, see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (1989); by Ausubel et al., Eds. CurrentProtocols in Molecular Biology, Current Protocols Press, (1994); and byBerger and Kimmel, Methods in Enzymology: Guide to Molecular CloningTechniques, Vol. 152, Academic Press, Inc., San Diego, Calif., (1987),the disclosures of which are hereby incorporated by reference.

[0058] In preferred aspects, the present invention employs yeast-E. colishuttle vectors to generate the constructs for the production ofknockout mice. Those of skill in the art will understand that theshuttle vector may be any yeast vector that is amenable for use inhomologous recombination. As such, it is contemplated that shuttlevectors such as, pRS416 and pRS426 (Sikorski et al., Genetics, 122:19-27(1989); Christian et al., Gene, 110:119-122, incorporated herein byreference) will be useful starting vectors for the construction of theshuttle vectors of the present invention.

[0059] In preparing the mouse genomic libraries, the vectors are firstrestriction digested to eliminate vector background. The digested vectoris then dephosphorylated in order to decrease the likelihood of vectorself ligation. The dephosphorylated vectors are then mixed with andligated to the size fractionated, Sau3A digested mouse genomic fragmentsto produce a vector comprising the mouse genomic DNA.

[0060] The shuttle vectors of the invention require an origin ofreplication functional in yeast and also an origin of replicationfunctional in bacteria. Yeast replication origins include Cen, 2 m andautonomous replication sequence (ARS). Preferably, the origin is a 2 morigin. Replication origins functional in bacteria are well known (e.g.,ColE1, F, or R1 based origins) and may give low or high copy numbers. Apreferred origin of replication functional in bacteria is a ColE1-typesuch as that present on plasmid pBR322. Where marker genes are employedin the vectors, depending on the nature of the marker gene, the markermay comprise transcriptional regulatory regions, particularly initiationregulatory regions.

[0061] Promoters and enhancers that will be useful regulatory regions insuch a context are well known to those of skill in the art and includebut are not limited to the HSV thymidine kinase (TK) promoter, humancytomegalovirus (CMV) immediate early gene promoter, the SV40 earlypromoter, the Rous sarcoma virus long terminal repeat promoter, β-actinpromoter, rat insulin promoter, the phosphoglycerol kinase promoter andglyceraldehyde-3-phosphate dehydrogenase promoter, all of which arepromoters well known and readily available to those of skill in the art,can be used to obtain high-level expression of the coding sequence ofinterest.

[0062] Inducible or regulatable promoters also are specificallycontemplated, such as, for example, those that are hormone or cytokineregulatable. Hormone regulatable promoters include MMTV, MT-1, ecdysoneand RuBisco as well as other hormone regulated promoters such as thoseresponsive to thyroid, pituitary and adrenal hormones. By employing apromoter with well known properties, the level and pattern of expressionof the protein of interest following transfection or transformation canbe optimized. Enhancers are organized much like promoters. That is, theyare composed of many individual elements, each of which binds to one ormore transcriptional proteins. The basic distinction between enhancersand promoters is operational. An enhancer region as a whole must be ableto stimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization. Enhancers useful in the present invention are well knownto those of skill in the art and will depend on the particularexpression system being employed (Scharf D et al (1994) Results ProblCell Differ 20: 125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).

[0063] In addition to the shuttle vector comprising the genomic library,a specific recombination cassette also is generated by using PCR. Such acassette generally may contain markers for selection in yeast and in EScells. In specific embodiments, the recombination cassette comprises theneomycin gene under the PGK promoter for ES selection and yeastnutritional marker URA3 gene provides for selection in yeast cells. The“Neo/Ura3” cassette-containing plasmid, pYYL-2 provides the universaltemplate for creating knockout recombinant cassettes for any gene ofinterest by PCR amplification. After PCR, the neo/ura3 cassette isflanked on each side by about 40 base pairs of unique sequencescorresponding to the region of the gene to be disrupted and throughwhich homologous recombination will occur. The selection of DNA sequencefor use as unique flanking sequences may be made based on the cDNAsequence of the gene or locus to be targeted or the genomic sequence butdoes not require the presence of known restriction sites nor does itrequire that restriction sites be engineered into the unique genomicDNA. It should be noted that the recombination cassette may alsocomprise a single marker sequence which allows selection in both yeastand ES cells. The length of the unique flanking sequences may vary fromabout 10 to 200 base pairs or more. Preferred lengths are from about 40to about 200 base pairs although longer sequences may increase theefficiency of recombination. Lengths of 1 kb may be advantageous to theefficiency of homologous recombination.

[0064] Other exemplary selectable markers include genes conferringresistance to hygromycin, genes encoding the Salmonella his D gene(which allows a cell to convert histidinal to histidine), puromycinD-acetyl transferase and others. The markers used are not limited tothose disclosed above but also include a variety of other selectablemarkers well known in the art and which are useful for selection inyeast, E. coli, and/or mammalian cells.

[0065] The DNA of the pool containing the shuttle vector, or theindividual vector, comprising all or part of the genomic region to betargeted and the recombination cassette are introduced into a yeaststrain by, for example, lithium acetate transformation or byelectroporation (or by other methods known in the art) eithersequentially or simultaneously.

[0066] Once in the yeast cell, the recombination cassette and theshuttle vector can recombine by homologous recombination via theirhomologous gene sequences (i.e., the flanking sequences of therecombination cassette and the genomic sequence in the shuttle vector),thereby inserting, into the target DNA sequence in the shuttle vector,the marker or markers from the recombination cassette thereby generatinga targeting construct. The targeting construct sequences will containintegrated markers that can be used in the selection of therecombination cassette flanked by sequences of the targeted gene byapplying selection for the yeast marker used.

[0067] In preferred embodiments, the yeast strain YLU-100 (Mata ura3-52lys2-801 ade2-101 trpl-D63 his3-D200, leu2-D1 Dsec71::His3) was used togenerate the mouse knock-out vector. In order to determine if homologousrecombination has occurred, the yeast is grown on media that will allowfor selection according to the markers employed in the shuttle vectorand the recombination cassette. If serial transformation is used, firstthe yeast is transformed with the DNA of the genomic clone or the poolcontaining the desired genomic clone and selected on growth medium thatwill allow for selection of using the yeast marker in the shuttlevector, e.g., growth on trp-deficient growth medium where the shuttlevector marker is TRP1. Subsequent transformation with the targetrecombination cassette and its successful recombination with the targetgenomic fragment is selected on the trp-deficient and ura-deficientgrowth medium where the yeast marker in the recombinant cassette isURA3. Alternatively, if cotransformation of genomic clone or poolcontaining the genomic clone is performed along with the recombinationcassette, the yeast containing the recombinant mouse knock-out targetingvector is selected on trp- and ura-deficient growth medium.

[0068] To confirm that the integration of the recombination cassetteoccurred by homologous recombination as opposed to some randomintegration, targeted plasmids may then analyzed by PCR using a primerfrom each side of the insertion. Finally, the targeting vectorcontaining the insertion is shuttled into bacteria so that adequatequantities of purified construct (e.g., plasmid) DNA can be prepared forfinal analysis and introduction into ES cells. In this way, targetingvectors can be generated with considerable ease and speed, obviating theextensive gene mapping and the search for suitable restriction sitesrequired by traditional methods. It should be noted that the ease ofconstruction and selection of targeting vectors according to the methodsof the present invention readily lends itself to automated procedures,particularly when certain physically detectable (e.g., calorimetric,fluorometric, and others) markers are used for selection of thetargeting vector.

[0069] II. Mouse Genomic Library Preparation and Amplification

[0070] The shuttle vectors of the present invention comprise genomic DNAfragments generated from mice. The genomic DNA is isolated according tomethods well known to those of skill in the art and may be derived fromany animal that is to be used to create a transgenic model. (Sambrook etal., 1989 Molecular Cloning: A laboratory manual, second edition). Inpreferred embodiments, the genomic DNA is derived from the 129svj mousestrain. Mouse genomic DNA derived from other mouse strains such as 129Ola, 129sv, C57BL/6 also may be used for the purpose of making mousegenomic library.

[0071] The shuttle vectors of the present invention comprise genomic DNAfragments; those of skill in the art will know various techniques forgenerating and separating such fragments. For example, purifiedhigh-molecular-weight genomic DNA is partially digested with arestriction enzyme such as Sau3A to randomly fragment the DNA and toobtain directly clonable ends. Other enzymes useful for this endeavorwill be well known to those of skill in the art. This randomlyfragmented DNA is size fractionated through, for example, sucrosedensity gradient. (Maniatis, T. et al. (1978). The isolation ofstructural genes from libraries of eukaryotic DNA. Cell 15: 687).

[0072] Preferred embodiments disclose a genomic library in which shuttlevectors comprise genomic fragments ranging from about 8 kb to about 13kb. This is merely an exemplary fragment range and fragments that arelarger or smaller than this also may be useful. Preferred embodimentsmay use shuttle vectors comprising a genomic fragment that is 1 kb, 2kb, 3 kb, 4 kb, 5kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 1 kb, 12 kb, 13 kb,14 kb, 15 kb, 16 kb, 17 kb, 18 kb or larger. In preferred embodiments,the genomic fragment inserts had an average insert size of 11 kb.

[0073] Methods for preparing genomic libraries and cDNA libraries arewell known in the art and are described in Sambrook et al., MolecularCloning, A Laboratory Manual, (section 9, pp. 9.2-9.58 and section 8,pp. 8.2-8.79, respectively) Cold Spring Harbor N.Y. (1989) and inCurrent Protocols in Molecular Biology, (section 5, pages 5.0.1-5.11.2)Ausubel et al., Eds. John Wiley and Sons Inc. (1987), the relevantsections of which are incorporated herein by reference.

[0074] In preferred aspects of the invention, the mouse genomic fragmentlibrary in the shuttle vector construct is amplified by transforming itinto electrocompetent bacterial cells. The electrocompetent bacterialcells that may be employed in the present invention include but are notlimited to eubacteria such as Gram-negative or Gram-positive organisms(e.g., E. coli (HB101, DH5a, DH10B and MC1061); Bacilli such as B.subtilis; Pseudomonas species, such as P. aeruginosa; Streptomyces spp.;Salmonella typhimurium; or Serratia marcescans.

[0075] In preferred embodiments, the shuttle vectors are transformedinto electrocompetent E. coli, ElectroMAX DH10B (GIBCO-BRL,Gaithersburg, Md.). A portion of each transformation is plated out and48 individual colonies from each are analyzed for desired size range ofinserts and percentage of clones containing inserts.

[0076] The amplified DNA and the E. coli containing the genomic mouseDNA inserts is organized into a library in which, for example, 1.5million independent clones generated from the cloning of the shuttlevector comprising the genomic DNA into the bacterial cells are dividedinto 300 pools of 5000 clones each and plated on 150 mm LB-Amp plates.The E. coli colonies are grown overnight and collected by scraping. Aportion of each of these pools is saved in, for example, three 96-wellmicrotiter plates and frozen at −70° C. The remainder is processed toobtain DNA, which is also saved in three 96-well microtiter plates. Thisorganization of the library format allows easy identification of pool/scontaining gene(s) of interest.

[0077] The appropriate pools containing gene or genes of interest fromthe mouse genomic library are identified by PCR amplification. PCRmethods are well known to those of skill in the art and are generallyset forth in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989);by Ausubel et al., eds Current Protocols in Molecular Biology, CurrentProtocols Press, (1994).

[0078] DNA from either each column of 8 pools or each row of 12 poolsfrom a 96 well plate is combined into a larger pool and assayed by PCRamplification using a pair of primers specific to the gene of interest.Primers are chosen such that they ideally amplify between 0.3 kb to 1.5kb fragment from the target area in the gene of interest. In designingthe primers mouse genomic sequence information of the gene of interestor the information from the human counterpart or closest family memberis used as a basis to estimate intron-exon structure of the gene ofinterest. However, this information is not essential. If genomicstructure is neither known nor can be estimated, then primers are chosenbased on the cDNA sequence to produce a product within the targeted areaof disruption. After identifying positive row(s) or column(s) ofcombined pools using this PCR step, positive combined pools are analyzedusing the same primers to identify the positive individual pool(s). Oncethe positive pools containing the gene of interest are identified, theyare used in homologous recombination to construct a knockout vector.

[0079] The inventors have found that successful recombination occurredusing pools containing up to 5000 clones. However, a linear decrease innumber of recombinants obtained occurred with an increase in poolcomplexity. A successful recombination event between the gene ofinterest and the neo/ura cassette depends on several factors. Theseinclude transformation efficiency of the host yeast strain, presence ofboth the genomic clone of interest and the neo/ura cassette within thesame yeast cell, and recombination frequency between the two constructsonce they are in the same cell.

[0080] III. Methods of Making Transgenic Animals

[0081] In order to introduce the targeting construct into the germlineof an animal, the targeting construct is first introduced into anundifferentiated totipotent cell termed an embryonic stem (ES) cellwherein the construct can recombine with the selected genomic region viatheir homologous sequences. ES cells are derived from an embryo orblastocyst of the same species as the developing embryo into which theyare to be introduced. ES cells are typically selected for their abilityto integrate into the inner cell mass and contribute to the germ line ofan individual when introduced into the mammal in an embryo at theblastocyst stage of development. Thus, any ES cell line having thiscapability is suitable for use in the practice of the present invention.

[0082] The cells are cultured and prepared for introduction of thetargeting construct using methods well known to the skilled artisan.(See, e.g., Robertson, E.J. ed. “Teratocarcinomas and Embryonic StemCells, a Practical Approach”, IRL Press, Washington D.C. (1987); Bradleyet al., Current Topics in Devel. Biol. 20:357-371 (1986); by Hogan etal. in “Manipulating the Mouse Embryo”: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. (1986); Thomas et al.,Cell, 51:503 (1987); Koller et al., Proc. Natl. Acad. Sci. USA, 88:10730(1991); Dorin et al., Transgenic Res., 1:101 (1992); and Veis et al.,Cell, 75:229 (1993) all of which are incorporated herein by reference).The targeting construct may be introduced into ES cells by any one ofseveral methods known in the art including electroporation, calciumphosphate co-precipitation, retroviral infection, microinjection,lipofection and other methods. Insertion of the targeting construct intothe targeted gene is typically detected by selecting cells forexpression of the marker gene contained in the targeting construct whichis typically under the control of a promoter which is functional in thetarget cell type (i.e., promoters which function in embryonic stemcells). ES cells expressing the marker sequence are then isolated andexpanded.

[0083] The ES cells having the disruption are then introduced into anearly-stage mouse embryo (e.g., blastocyst) (see, e.g., Robertson,supra, Bradley, supra, and Monsour et al., Nature, 336:348 (1988))incorporated herein by reference. Blastocysts and other early stageembryos used for this purpose are obtained by flushing the uterus ofpregnant animals for example, by the methods described in Robertson etal., supra and Bradley et al., supra. The suitable stage of developmentfor the blastocyst is species dependent; however, for mice it is about3.5 days post-fertilization.

[0084] While any embryo of the right age/stage of development issuitable for implantation of the modified ES cell, preferred mostembryos are male and have genes coding for a coat color or otherphenotypic marker that is different from the coat color or otherphenotypic marker encoded by the ES cell genes. In this way, theoffspring can be screened easily for the presence of the targetedmutation by looking for mosaic coat color (e.g. agouti) or the otherphenotypic markers (indicating that the ES cell was incorporated intothe developing embryo). Thus, for example, if the ES cell line carriesthe genes for white fur, the host embryos selected will preferably carrygenes for black or agouti fur.

[0085] An alternate method of preparing an embryo containing ES cellsthat possess the targeting construct is to generate “aggregationchimeras”. A morula of the proper developmental stage (about 2½ dayspost-fertilization for mice) is isolated. The zona pellucida can beremoved by treating the morula with a solution of mild acid for about 30seconds, thereby exposing the “clump” of cells that comprise the morula.Certain types of ES cells such as the RI cell line for mice can then beco-cultured with the morula cells, forming an aggregation chimera embryoof morula and ES cells, (Joyner, A.L., “Gene Targeting”, The PracticalApproach Series, JRL Press Oxford University Press, New York, 1993,incorporated herein by reference).

[0086] A refinement of the aggregation chimera embryo method can be usedto generate an embryo comprised of essentially only those ES cellscontaining the knockout construct. In this technique, a very early stagezygote (e.g., a two-cell stage zygote for mice) is given a mild electricshock. This shock serves to fuse the nuclei of the cells in the zygotethereby generating a single nucleus that has two-fold (or more) the DNAof a naturally occurring zygote of the same developmental stage. Thesezygotic cells are excluded from the developing embryo proper, andcontribute only to forming accessory embryonic structures such as theextra-embryonic membrane. Therefore, when ES cells are co-cultured withthe zygotic cells, the developing embryo is comprised exclusively of EScells, (see Joyner, A.L., supra).

[0087] After the ES cells have been incorporated into the aggregationchimera or into the blastocyst, the embryos may be implanted into theuterus of a pseudo pregnant foster mother. While any foster mother maybe used, preferred foster mothers are typically selected for theirability to breed and reproduce well, and for their ability to care fortheir young. Such foster mothers are typically prepared by mating withvasectomized males of the same species. The pseudo pregnant stage of thefoster mother is important for successful implantation, and it isspecies dependent. For mice, this stage is about 2-3 days ofpseudopregnancy.

[0088] Offspring that are born to the foster mother may be screenedinitially for mosaic coat color or another phenotypic marker (where thephenotype selection strategy has been employed). In addition, or as analternative, chromosomal DNA obtained from tail tissue of the offspringmay be screened for the presence of the targeted mutation using Southernblots and/or PCR. The offspring that are positive for homologousrecombination at the targeted locus will typically be a mosaic ofwild-type cells derived from the host embryo and heterozygous cellsderived from injected ES cells (i.e., chimeric offspring). Chimericoffspring are crossed with wild-type partners to generate offspring thatare heterozygous for the targeted mutations, i.e., all of their cellsare heterozygous for the mutation.

[0089] Methods for producing transgenic mammals, including rabbits,pigs, and rats, using micro-injection are described in Hamer et al,Nature 315:680-683 (1985); U.S. Pat. No. 4,736,866, incorporated hereinby reference. Additional methods for producing transgenic animals aregenerally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191; whichis incorporated herein by reference), Brinster et al. 1985; which isincorporated herein by reference in its entirety) and in “Manipulatingthe Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan,Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press,1994; which is incorporated herein by reference in its entirety).Briefly, this method involves injecting DNA into a fertilized egg, orzygote, and then allowing the egg to develop in a pseudo-pregnantmother. The zygote can be obtained using male and female animals of thesame strain or from male and female animals of different strains. Thetransgenic animal that is born, the founder, is bred to produce moreanimals with the same DNA insertion. In this method of making transgenicanimals, the new DNA typically randomly integrates into the genome by anon-homologous recombination event. One to many thousands of copies ofthe DNA may integrate at one site in the genome.

[0090] Generally, the DNA is injected into one of the pronuclei, usuallythe larger male pronucleus. The zygotes are then either transferred thesame day, or cultured overnight to form 2-cell embryos and thentransferred into the oviducts of pseudo-pregnant females. The animalsborn are screened for the presence of the desired integrated DNA.

[0091] DNA clones for microinjection can be prepared by any means knownin the art. For example, DNA clones for microinjection can be cleavedwith enzymes appropriate for removing the bacterial plasmid sequences,and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 mg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA.

[0092] Additional methods for purification of DNA for microinjection aredescribed in Hogan et al. Manipulating the Mouse Embryo (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986), in Palmiter et al.Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rdedition, published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrooket al. Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989).

[0093] In an exemplary microinjection procedure, female mice six weeksof age are induced to superovulate. The superovulating females areplaced with males and allowed to mate. After approximately 21 hours, themated females are sacrificed and embryos are recovered from excisedoviducts and placed in an appropriate buffer, e.g., Dulbecco's phosphatebuffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surroundingcumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclearembryos are then washed and placed in Earle's balanced salt solutioncontaining 0.5% BSA in a 37.5° C. incubator with a humidified atmosphereat 5% CO₂, 95% air until the time of injection. Embryos can be implantedat the two-cell stage.

[0094] Randomly cycling adult female mice are paired with vasectomizedmales. C57BL/6 or Swiss mice or other comparable strains can be used forthis purpose. Recipient females are mated at the same time as donorfemales. At the time of embryo transfer, the recipient females areanesthetized with an intraperitoneal injection of 0.015 ml of 2.5%avertin per gram of body weight. The oviducts are exposed by a singlemidline dorsal incision. An incision is then made through the body walldirectly over the oviduct. The ovarian bursa is then torn withwatchmakers forceps. Embryos to be transferred are placed in DPBS(Dulbecco's phosphate buffered saline) and in the tip of a transferpipette (about 10 to 12 embryos). The pipette tip is inserted into theinfundibulum and the embryos transferred. After the transfer, theincision is closed by two sutures. The pregnant animals then give birthto the founder animals which are used to establish the transgenic line.

[0095] If animals homozygous for the targeted mutation are desired, theycan be prepared by crossing animals heterozygous for the targetedmutation. Mammals homozygous for the disruption may be identified bySouthern blotting of equivalent amounts of genomic DNA from mammals thatare the product of this cross, as well as mammals of the same speciesthat are known heterozygotes, and wild-type mammals. Alternatively,specific restriction fragment length polymorphisms can be detected whichco-segregate with the mutant locus. Probes to screen the Southern blotsfor the presence of the targeting construct in the genomic DNA can bedesigned as described below.

[0096] Other means of identifying and characterizing the offspringhaving a disrupted gene are also available. For example, Northern blotscan be used to probe mRNA obtained from various tissues of the offspringfor the presence or absence of transcripts. Differences in the length ofthe transcripts encoded by the targeted gene can also be detected. Inaddition, Western blots can be used to assess the level of expression ofthe targeted gene by probing the Western blot with an antibody againstthe protein encoded by the targeted gene. Protein for the Western blotmay be isolated from tissues where this gene is normally expressed.Finally, in situ analysis (such as fixing the cells and labeling withantibody or nucleic acid probe) and/or FACS (fluorescence activated cellsorting) analysis of various cells from the offspring can be conductedusing suitable antibodies to look for the presence or absence of thegene product.

IV. EXAMPLES

[0097] The present invention is described in more detail with referenceto the following non-limiting examples which represent preferredembodiments of the invention. Those of skill in the art will understandthat the techniques described in these examples represent techniquesdescribed by the inventors to function well in the practice of theinvention, and as such constitute preferred modes for the practicethereof. However, it should be appreciated that those of skill in theart should in light of the present disclosure, appreciate that manychanges can be made in the specific methods which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Example 1 Construction of the Shuttle Vector

[0098] The present example provides construction and description of theyeast-E. coli shuttle vector pYYL-1 employed in the present invention.(See FIG. 4). The base vector for construction of pYYL-1 wasyeast-E-coli shuttle vector pGBT9 (Genbank Accession #U07646) pGBT9 wasrestriction digested with SphI and the 4.4 kb vector fragment wasisolated. Oligonucleotides of SEQ ID NO:1 and SEQ ID NO:2 were annealedand ligated with the 4.4 kb vector fragment at the SphI sites. Theannealed oligonucleotide contains SP6 and T7 primer sites and apolylinker containing several unique cloning sites. The resultingligated vector is named pYYL-1.

[0099] SEQ ID NO:1 CAT TTA GGT GAC ACT ATA GCG GCC GCG GAT CCC TAT AGTGAG TCG TAT TAC GGA CCG TCG ACT TAA TTA ACA TG

[0100] SEQ ID NO:2 TTA ATT AAG TCG ACG GTC CGT AAT ACG ACT CAC TAT AGGGAT CCG CGG CCG CTA TAG TGT CAC CTA AAT GCA TG

[0101] The sequence of the pYYL-1 vector is provided in SEQ ID NO:3.This plasmid is an E. coli-yeast shuttle vector that contains a B1a1gene (for ampicillin resistance) and Co1E1-ORI (replication origin) formaintenance and propagation in E. coli; a 2 m origin of replication andTRP1 gene for propagation in yeast; and TK gene for negative selectionin mammalian cells.

[0102] The unique BamHI site in this vector is used for inserting thegenomic library fragments. The vector was first restriction digested tocompletion with BamHI to completion and then dephosphorylated using CalfIntestinal Alkaline Phosphtase (New England Biolabs, Beverly, Mass.).Subsequently, Sau3A digested mouse genomic DNA, containingphosphorylated ends, was ligated with the treated vectors. The BamHIsite in pYYL-1 site is flanked by Sp6 and T7 priming sequences which canbe used in PCR analysis of the inserted fragment in the steps before andafter recombination. To facilitate excision of the genomic fragmentalong with the negative selection marker TK, rare cutting enzyme sitesflanking these regions have been engineered into pYYL-1. These sites areNotI on one end and PacI and SalI on the end closer to TK. Of course itshould be understood that the rare cutting enzyme sites, markers andflanking sites are only exemplary. Other vectors also may be constructedhaving alternative replication origins, selection genes and restrictionsites, which would be equally useful for the purpose of making mousegenomic libraries and subsequently used in constructing mouse knockoutvectors by yeast recombination.

EXAMPLE 2 Construction of the Recombination Cassette

[0103] For easy generation of the recombination cassette, the neomycingene under the PGK promoter and yeast nutritional marker URA3 gene havebeen cloned in tandem into the pSPORT-1 vector (GIBCO-BRL, Gaithersburg,Md.). A Smal-HindIII fragment containing the yeast URA3 selection markerwas excised from YEP24 plasmid (ATCC#37051) and cloned into pSPORT1 atthe corresponding sites. Into this plasmid, an AscI fragment containingthe Neomycin gene from pKONeo (Stratagene, La Jolla, Calif.) was clonedat the Mlu I site. The resultant Neo/Ura3 cassette containing plasmid,pYYL-2, provides the universal template for creating knockoutrecombinant cassettes for any gene of interest by PCR amplification.

Example 3 Construction of Mouse Knock-out Vector for GPR-24 Gene

[0104] GPR-24 (also called SLC-1) is a recently discovered Gprotein-coupled receptor, which is homologous to somatostatin receptors,and binds to the melanin-concentrating hormone ( MCH) (Saito et al.1999. Nature 400:265-269). The coding region of this gene has one 1115bp intron (intron I).

[0105] Two primers (SEQ ID NO:4 and SEQ ID NO:5), which amplified anapproximately 2.2 kb fragment spanning the initiating Met and stop codonand contained the intron I, sequence were used for identification ofpositive pools of the arrayed mouse genomic library. PCR were performedon DNA from 24 combined pools from three 96-well plates. Each combinedpool was comprised of DNA pooled from the 12 wells of each row. Thepositive pools gave a PCR product of approximately 2.2 kb. PCRamplification was repeated on individual pools comprising two of thepositive combined pools. Once the individual positive pools were found,PCR was performed on the positive pools using the gene-specific primersand the vector-specific primers to determine that the desired positiveclone had sufficient flanking genomic sequence outside the targetedregion of recombination. Each gene specific primer (SEQ ID NO:4 and SEQID NO:5) was paired with both vector specific primers, namely Sp6 and T7(SEQ ID NO:6 and SEQ ID NO:7) to perform the PCR. A positive individualpool that had one long arm of approximately 11.7 kb and a short arm ofapproximately 1.3 kb flanking the targeted recombination region was usedin the next recombination step.

[0106] To generate the recombination cassette, two (sense and antisense)oligonucleotide primers were synthesized of which 45 nucleotides of eachwere homologous to the targeted area of GPR-24. In addition to thehomology to the target region, the sense primer at its 3′ end alsocontains additional 20 bp that correspond to the 5′ end of the neomycingene (SEQ ID NO:8). In the antisense primer 25 bp at the 3′ end arehomologous to the 3′ end of the URA3 gene (SEQ ID NO:9). Using theseprimers, the recombination cassette for GPR-24 was generated, whichincluded 45-bp flanking sequences at its ends homologous to GPR-24.These primers were designed to delete sequence by recombination fromexon I and exon II corresponding to amino terminal 128 amino acids andintron I and replace it with the neo/ura selection cassette (see FIG.5).

[0107] Yeast transformation was performed sequentially. 2.0 mg DNA ofthe positive pool (in which the desired gene is present in approximately1:5000 ratio) or from an array in which the positive clone was mixedamong other clones in ratios of 1:250, 1:500, and 1:1000 was transformedinto yeast and plated onto trp-deficient selective medium. Approximately20,000 yeast colonies from transformation plates were scraped and thecells were made competent. 100 ml (2 OD₆₀₀ cells) were used to transform1 mg of cassette DNA. Transformation mixtures were plated on trp- andura-deficient selective growth medium. In each case, yeast colonies wereobtained; however, the number of Trp+Ura+colonies decreased with thecomplexity of the pool.

[0108] The presence of recombined GPR24 knockout construct in yeastcolonies was confirmed by PCR. The gene-specific primer (SEQ ID NO:10)paired with the cassette-specific neo primer (SEQ ID NO:12) and the genespecific primer (SEQ ID NO:11) paired with the cassette-specific uraprimer (SEQ ID NO:13), respectively, produced 388 bp and 536 bpfragments, thus confirming homologous recombination at the targetedsite. In addition, PCR was performed again to confirm the length andshort arms. Positive yeast colonies were grown and plasmids rescued,amplified, and transfected into mouse ES cells.

Example 4 Construction of Mouse Knockout Vector for CHL-1 Gene

[0109] CHL-1 is a novel chordin-like secreted molecule. Methods andcompositions directed to this gene are disclosed in No. 60/169,494 filedDec. 7, 1999 (incorporated herein by reference). The knockout constructfor the mouse CHL-1 was constructed as follows: a sense primer specificto signal peptide coding exon (SEQ ID NO:14) and an antisense primer(SEQ ID NO:15) in the adjacent intron downstream of the signal peptidewere used to generate a 405-bp PCR product in positive pools. Individualgenomic clones of CHL-1 were isolated by colony hybridization. Thepositive clone that contained an approximately 9.2 kb fragment was usedfor recombination. The neo/ura cassette flanked with 45 bp CHL-1specific targeting sequences was generated by PCR amplification usingprimers of SEQ ID NO:16 and SEQ ID NO:17. The cassette was designed todelete 59 bp from exon I, which includes sequence encoding theinitiating methionine and signal peptide (see FIG. 6).

[0110] 200 ng of the CHL-1 genomic clone DNA and 500 ng of the neo/urarecombination cassette were co-transformed into the yeast competentcells. Under these conditions, 53 yeast colonies grew on trp- andura-deficient synthetic medium plates. 8 of these colonies were used toconfirm recombination of the cassette with the genomic clone by PCR. Thegene specific primer (SEQ ID NO:18) paired with the cassette specificneo primer (SEQ ID NO:12) and the gene-specific primer (SEQ ID NO:19)paired with the cassette-specific ura primer (SEQ ID NO:13),respectively produced 500-bp and 693-bp fragments, which indicatedsuccessful recombination. The plasmid-specific primers Sp6 or T7 wereused in combination with the cassette-specific neo or ura primers toestimate length of flanking genomic sequences to the neo/ura cassette.The analysis showed that the long arm was approximately 6.6 kb and theshort arm was approximately 2.5 kb. Plasmids were rescued and processedas described above for GPR24.

[0111] While the methods and compositions herein have been described interms of preferred embodiments, it will be apparent that variations maybe applied to the methods and/or compositions without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that assays which are physiologically related may besubstituted for the assays described herein while still producing thesame or similar results. All such similar substitutes and modificationsapparent to those of skill in the art are deemed to be within the scopeof the invention as defined by the appended claims.

[0112] To the extent that certain exemplary procedural or other detailssupplementary to those described herein may be found in the referencescited herein, such references are all specifically incorporated hereinby reference.

1 19 1 74 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 catttaggtg acactatagc ggccgcggat ccctatagtg agtcgtattacggaccgtcg 60 acttaattaa catg 74 2 19 DNA Artificial SequenceDescription of Artificial Sequence primer 2 gagcagttgg gctcagagg 19 3 22DNA Artificial Sequence Description of Artificial Sequence vectorsequence 3 gtgatgctaa tgaacgagag ag 22 4 21 DNA Artificial SequenceDescription of Artificial Sequence primer 4 gtgctacttc catttgtcac g 21 521 DNA Artificial Sequence Description of Artificial Sequence primer 5agcagaattg tcatgcaagg g 21 6 20 DNA Artificial Sequence Description ofArtificial Sequence primer 6 gtagaagatg gatggcatga 20 7 22 DNAArtificial Sequence Description of Artificial Sequence primer 7cagctcatct gtcagatatt tc 22 8 65 DNA Artificial Sequence Description ofArtificial Sequence primer 8 ccttggagcc cctgaattgc attttgcagt agctcgaaggagaaacaagg cagtctggag 60 catgc 65 9 70 DNA Artificial SequenceDescription of Artificial Sequence primer 9 tggaagacac ttacgttttacttgttctgt tttgcttcct tctaggtgag tttagtatac 60 atgcatttac 70 10 21 DNAArtificial Sequence Description of Artificial Sequence primer 10ggtctcagtg ttggaacagg a 21 11 21 DNA Artificial Sequence Description ofArtificial Sequence primer 11 gtacagttga gaagaggaaa g 21 12 74 DNAArtificial Sequence Description of Artificial Sequence primer 12ttaattaagt cgacggtccg taatacgact cactataggg atccgcggcc gctatagtgt 60cacctaaatg catg 74 13 6515 DNA Artificial Sequence Description ofArtificial Sequence primer 13 gcttgcatgc atttaggtga cactatagcggccgcggatc cctatagtga gtcgtattac 60 ggaccgtaga gtcgagcagt gtggttttcaagaggaagca aaaagcctct ccacccaggc 120 ctggaatgtt tccacccaat gtcgagcagtgtggttttgc aagaggaagc aaaaagcctc 180 tccacccagg cctggaatgt ttccacccaatgtcgagcaa accccgccca gcgtcttgtc 240 attggcgaat tggaacacgc agatgcagtcggggcggcgc ggtcccaggt ccacttcgca 300 tattaaggtg acgcgtgtgg cctcgaacaccgagcgaccc tgcagcgacc cgcttaacag 360 cgtcaacagc gtgccgcaca tcttggtggcgtgaaactcc cgcacctctt cggccagcgc 420 cttgtagaag cgcgtatggc ttcgtaccccggccatcagc acgcgtctgc gttcgaccag 480 gctgcgcgtt ctcgcggcca tagcaaccgacgtacggcgt tgcgccctcg ccggcagcaa 540 gaagccacgg aagtccgccc ggagcagaaaatgcccacgc tactgcgggt ttatatagac 600 ggtccccacg ggatggggaa aaccaccaccacgcaactgc tggtggccct gggttcgcgc 660 gacgatatcg tctacgtacc cgagccgatgacttactggc gggtgctggg ggcttccgag 720 acaatcgcga acatctacac cacacaacaccgccttgacc agggtgagat atcggccggg 780 gacgcggcgg tggtaatgac aagcgcccagataacaatgg gcatgcctta tgccgtgacc 840 gacgccgttc tggctcctca tatcgggggggaggctggga gctcacatgc cccgcccccg 900 gccctcaccc tcatcttcga ccgccatcccatcgccgcct tcctgtgcta cccggccgcg 960 cgatacctta tgggcagcat gaccccccaggccgtgctgg cgttcgtggc cctcatcccg 1020 ccgaccttgc ccggcacaaa catcgtgttgggggcccttc cggaggacag acacatcgac 1080 cgcctggcca aacgccagcg ccccggcgagcggcttgacc tggctatgct ggccgcgatt 1140 cgccgcgttt acgggctgct tgccaatacggtgcggtatc tgcagggcgg cgggtcgtgg 1200 cgggaggatt ggggacagct ttcggggacggccgtgccgc cccagggtgc cgagccccag 1260 agcaacgcgg gcccacgacc ccatatcggggacacgttat ttaccctgtt tcgggccccc 1320 gagttgctgg cccccaacgg cgacctgtacaacgtgtttg cctgggcctt ggacgtcttg 1380 gccaaacgcc tccgtcccat gcacgtctttatcctggatt acgaccaatc gcccgccggc 1440 tgccgggacg ccctgctgca acttacctccgggatgatcc agacccacgt caccacccca 1500 ggctccatac cgacgatctg cgacctggcgcgcacgtttg cacgggagat gggggaggct 1560 aactgaaaca cggaaggaga caataccggaaggaacctgc gctatgacgg caataaaaag 1620 acagaataaa acgcacgggt gttgggtcgtttgttcataa acgcggggtt cggtcccagg 1680 gctggcactc tgtcgatacc ccaccgagaccccattgggg ccaatacgcc cgcgtttctt 1740 ccttttcccc accccacccc ccaagttcgggtgaaggccc agggctcgca gccaacgtcg 1800 gggcggcagg cctgccatag ccacgggccccgtgggttag ggacggggtc ccccatgggg 1860 aatggtttat ggttcgtggg ggttattattttgggcgttg cgtggggtca gtccacgact 1920 ggactgagca gacagaccca tggtttttggatggcctggg catggaccgc atgtactggc 1980 gcgacacgaa caccgggcgt ctgtggctgccaaacacccc cgacccccaa aaaccaccgc 2040 gcggatttct ggcgccgccg gacgaactaaacctgactac ggaccgtcga cttaattaac 2100 atgccggcaa gtgcacaaac aatacttaaataaatactac tcagtaataa cctatttctt 2160 agcatttttg acgaaatttg ctattttgttagagtctttt acaccatttg tctccacacc 2220 tccgcttaca tcaacaccaa taacgccatttaatctaagc gcatcaccaa cattttctgg 2280 cgtcagtcca ccagctaaca taaaatgtaagctttcgggg ctctcttgcc ttccaaccca 2340 gtcagaaatc gagttccaat ccaaaagttcacctgtccca cctgcttctg aatcaaacaa 2400 gggaataaac gaatgaggtt tctgtgaagctgcactgagt agtatgttgc agtcttttgg 2460 aaatacgagt cttttaataa ctggcaaaccgaggaactct tggtattctt gccacgactc 2520 atctccatgc agttggacga tatcaatgccgtaatcattg accagagcca aaacatcctc 2580 cttaggttga ttacgaaaca cgccaaccaagtatttcgga gtgcctgaac tatttttata 2640 tgcttttaca agacttgaaa ttttccttgcaataaccggg tcaattgttc tctttctatt 2700 gggcacacat ataataccca gcaagtcagcatcggaatct agagcacatt ctgcggcctc 2760 tgtgctctgc aagccgcaaa ctttcaccaatggaccagaa ctacctgtga aattaataac 2820 agacatactc caagctgcct ttgtgtgcttaatcacgtat actcacgtgc tcaatagtca 2880 ccaatgccct ccctcttggc cctctccttttcttttttcg accgaattaa ttcgtaatca 2940 tggtcatagc tgtttcctgt gtgaaattgttatccgctca caattccaca caacatacga 3000 gccggaagca taaagtgtaa agcctggggtgcctaatgag tgaggtaact cacattaatt 3060 gcgttgcgct cactgcccgc tttccagtcgggaaacctgt cgtgccagct ggattaatga 3120 atcggccaac gcgcggggag aggcggtttgcgtattgggc gctcttccgc ttcctcgctc 3180 actgactcgc tgcgctcggt cgttcggctgcggcgagcgg tatcagctca ctcaaaggcg 3240 gtaatacggt tatccacaga atcaggggataacgcaggaa agaacatgtg agcaaaaggc 3300 cagcaaaagg ccaggaaccg taaaaaggccgcgttgctgg cgtttttcca taggctccgc 3360 ccccctgacg agcatcacaa aaatcgacgctcaagtcaga ggtggcgaaa cccgacagga 3420 ctataaagat accaggcgtt tccccctggaagctccctcg tgcgctctcc tgttccgacc 3480 ctgccgctta ccggatacct gtccgcctttctcccttcgg gaagcgtggc gctttctcat 3540 agctcacgct gtaggtatct cagttcggtgtaggtcgttc gctccaagct gggctgtgtg 3600 cacgaacccc ccgttcagcc cgaccgctgcgccttatccg gtaactatcg tcttgagtcc 3660 aacccggtaa gacacgactt atcgccactggcagcagcca ctggtaacag gattagcaga 3720 gcgaggtatg taggcggtgc tacagagttcttgaagtggt ggcctaacta cggctacact 3780 agaaggacag tatttggtat ctgcgctctgctgaagccag ttaccttcgg aaaaagagtt 3840 ggtagctctt gatccggcaa acaaaccaccgctggtagcg gtggtttttt tgtttgcaag 3900 cagcagatta cgcgcagaaa aaaaggatctcaagaagatc ctttgatctt ttctacgggg 3960 tctgacgctc agtggaacga aaactcacgttaagggattt tggtcatgag attatcaaaa 4020 aggatcttca cctagatcct tttaaattaaaaatgaagtt ttaaatcaat ctaaagtata 4080 tatgagtaaa cttggtctga cagttaccaatgcttaatca gtgaggcacc tatctcagcg 4140 atctgtctat ttcgttcatc catagttgcctgactccccg tcgtgtagat aactacgata 4200 cgggagggct taccatctgg ccccagtgctgcaatgatac cgcgagaccc acgctcaccg 4260 gctccagatt tatcagcaat aaaccagccagccggaaggg ccgagcgcag aagtggtcct 4320 gcaactttat ccgcctccat ccagtctattaattgttgcc gggaagctag agtaagtagt 4380 tcgccagtta atagtttgcg caacgttgttgccattgcta caggcatcgt ggtgtcacgc 4440 tcgtcgtttg gtatggcttc attcagctccggttcccaac gatcaaggcg agttacatga 4500 tcccccatgt tgtgcaaaaa agcggttagctccttcggtc ctccgatcgt tgtcagaagt 4560 aagttggccg cagtgttatc actcatggttatggcagcac tgcataattc tcttactgtc 4620 atgccatccg taagatgctt ttctgtgactggtgagtact caaccaagtc attctgagaa 4680 tagtgtatgc ggcgaccgag ttgctcttgcccggcgtcaa tacgggataa taccgcgcca 4740 catagcagaa ctttaaaagt gctcatcattggaaaacgtt cttcggggcg aaaactctca 4800 aggatcttac cgctgttgag atccagttcgatgtaaccca ctcgtgcacc caactgatct 4860 tcagcatctt ttactttcac cagcgtttctgggtgagcaa aaacaggaag gcaaaatgcc 4920 gcaaaaaagg gaataagggc gacacggaaatgttgaatac tcatactctt cctttttcaa 4980 tattattgaa gcatttatca gggttattgtctcatgagcg gatacatatt tgaatgtatt 5040 tagaaaaata aacaaatagg ggttccgcgcacatttcccc gaaaagtgcc acctgacgtc 5100 taagaaacca ttattatcat gacattaacctataaaaata ggcgtatcac gaggcccttt 5160 cgtctcgcgc gtttcggtga tgacggtgaaaacctctgac acatgcagct cccggagacg 5220 gtcacagctt gtctgtaagc ggatgccgggagcagacaag cccgtcaggg cgcgtcagcg 5280 ggtgttggcg ggtgtcgggg ctggcttaactatgcggcat cagagcagat tgtactgaga 5340 gtgcaccata acgcatttaa gcataaacacgcactatgcc gttcttctca tgtatatata 5400 tatacaggca acacgcagat ataggtgcgacgtgaacagt gagctgtatg tgcgcagctc 5460 gcgttgcatt ttcggaagcg ctcgttttcggaaacgcttt gaagttccta ttccgaagtt 5520 cctattctct agctagaaag tataggaacttcagagcgct tttgaaaacc aaaagcgctc 5580 tgaagacgca ctttcaaaaa accaaaaacgcaccggactg taacgagcta ctaaaatatt 5640 gcgaataccg cttccacaaa cattgctcaaaagtatctct ttgctatata tctctgtgct 5700 atatccctat ataacctacc catccacctttcgctccttg aacttgcatc taaactcgac 5760 ctctacattt tttatgttta tctctagtattactctttag acaaaaaaat tgtagtaaga 5820 actattcata gagtgaatcg aaaacaatacgaaaatgtaa acatttccta tacgtagtat 5880 atagagacaa aatagaagaa accgttcataattttctgac caatgaagaa tcatcaacgc 5940 tatcactttc tgttcacaaa gtatgcgcaatccacatcgg tatagaatat aatcggggat 6000 gcctttatct tgaaaaaatg cacccgcagcttcgctagta atcagtaaac gcgggaagtg 6060 gagtcaggct ttttttatgg aagagaaaatagacaccaaa gtagccttct tctaacctta 6120 acggacctac agtgcaaaaa gttatcaagagactgcatta tagagcgcac aaaggagaaa 6180 aaaagtaatc taagatgctt tgttagaaaaatagcgctct cgggatgcat ttttgtagaa 6240 caaaaaagaa gtatagattc tttgttggtaaaatagcgct ctcgcgttgc atttctgttc 6300 tgtaaaaatg cagctcagat tctttgtttgaaaaattagc gctctcgcgt tgcatttttg 6360 ttttacaaaa atgaagcaca gattcttcgttggtaaaata gcgctttcgc gttgcatttc 6420 tgttctgtaa aaatgcagct cagattctttgtttgaaaaa ttagcgctct cgcgttgcat 6480 ttttgttcta caaaatgaag cacagatgcttcgtt 6515 14 19 DNA Artificial Sequence Description of ArtificialSequence primer 14 ccggctgcat ggatctgca 19 15 21 DNA Artificial SequenceDescription of Artificial Sequence primer 15 agtccacgga aacgaaagac a 2116 21 DNA Artificial Sequence Description of Artificial Sequence primer16 cttgcatgca tttaggtgac a 21 17 21 DNA Artificial Sequence Descriptionof Artificial Sequence primer 17 gtaatacgac tcactatagg g 21 18 65 DNAArtificial Sequence Description of Artificial Sequence primer 18gtgggtggac gggcgctcca ctccagggag caggcgacct gcacccaagg cagtctggag 60catgc 65 19 70 DNA Artificial Sequence Description of ArtificialSequence primer 19 caagtagcgg tcaatggcca tagcagtcag gatgtaggtgctggtgtgag tttagtatac 60 atgcatttac 70

1. A method of preparing a genomic library for use in producing knockouttargeting vectors comprising: a) preparing genomic DNA fragments ofpre-selected sizes; b) preparing a shuttle vector comprising insertingsaid genomic DNA fragments into a yeast vector, wherein the vectorcomprises: i) a first bacterial origin of replication; ii) a firstbacterial selection marker; iii) a first yeast origin of replication;iv) a first yeast selection marker; and v) a first mammalian selectionmarker; c) introducing said vector into bacterial host cells to amplifysaid shuttle vectors in transformed bacterial host cells; and; d)arraying said transformed bacterial host cells into pools wherein thebacterial host cells comprise said shuttle vectors and wherein saidpools comprise genomic fragments of pre-selected sizes.
 2. The method ofclaim 1, wherein said shuttle vectors in said bacterial host cellscomprise: vi) a genomic DNA fragment; ii) a first yeast origin ofreplication; iii) a first yeast selection marker; iv) a first bacterialorigin of replication; v) a first bacterial selection marker; and vi) afirst selection marker for integration into mammalian cells.
 3. Themethod of claim 1, wherein said genomic DNA comprises mouse genomic DNAfragments ranging from about 8 kb to about 14 kb.
 4. The method of claim3, wherein said mouse genomic DNA fragments are isolated from a mousestrain selected from the group consisting of 129svj, 129 Ola, 129sv, andC57BL/6.
 5. The method of claim 1, wherein said genomic fragments ofstep (c) generate between about 3×10⁶ and 5×10⁶ clones.
 6. The method ofclaim 1, wherein said host cells are bacterial cells selected from thegroup consisting of Escherichia coli, Bacillus subtilis, Pseudomonasaeruginosa, Salmonella typhimurium and Serratia marcescans.
 7. Themethod of claim 6, wherein said host cell is E. coli.
 8. The method ofclaim 1, wherein said bacterial origin of replication is selected fromthe group consisting of ColE1-ORI, F and R1 based bacterial origin ofreplication.
 9. The method of claim 1, wherein said bacterial origin ofreplication is an E. coli origin of replication.
 10. The method of claim9, wherein said E. coli origin of replication is ColE1-ORI.
 11. Themethod of claim 1, wherein said yeast origin of replication is selectedfrom the group consisting of Cen, 2 m and the autonomous replicationsequence.
 12. The method of claim 1, wherein said marker for bacterialpropagation is selected from the group consisting of ampicillinresistance, tetracycline resistance, neomycin resistance, kanamycinresistance and chloramphenicol resistance.
 13. The method of claim 12,wherein said marker for ampicillin resistance is BlaI.
 14. The method ofclaim 1, wherein said marker for propagation in yeast is selected fromthe group consisting of trp1, His, Ura3, Arg, Ade and Leu2.
 15. Themethod of claim 1, wherein said selectable marker for mammalian cells isselected from the group consisting of neomycin resistance, hygromycinresistance, zeocin resistance, Salmonella HisD and puromycin-acetyltransferase.
 16. The method of claim 1, further comprising a negativeselectable marker.
 17. The method of claim 16, wherein said negativeselectable marker is selected from the group consisting of thymidinekinase, and xanthine-guanine-phosphoribosyltransferase.
 18. The methodof claim 1, wherein said yeast vector further comprises a BamHI site forinserting said genomic fragments.
 19. The method of claim 18, whereinsaid BamHI site is flanked by priming sequences to facilitate PCRamplification.
 20. The method of claim 19, wherein said primingsequences are Sp6 and T7 priming sequences.
 21. The method of claim 1,wherein said yeast vector is designated pYYL-1.
 22. The method of claim1, wherein said shuttle vector further comprises rare cutting enzymesites flanking the genomic fragment.
 23. The method of claim 17, whereinsaid shuttle vector comprises rare cutting enzyme sites flanking themammalian selection marker.
 24. A genomic library prepared according tothe method of claim
 1. 25. The genomic library of claim 24, wherein saidgenomic library is used for high throughput construction of knockoutvectors.
 26. The library of claim 24, wherein said genomic library isused for high throughput construction of mouse knockout vectors.
 27. Amethod of preparing a gene targeting vector for homologous recombinationcomprising: a) selecting a bacterial clone pool positive for the gene tobe targeted from an array of bacterial clones comprising the genomiclibrary said array prepared according to the method of claim 1; b)isolating the DNA from said positive pool; c) preparing a secondexpression construct comprising a marker cassette comprising a secondyeast selectable marker and a second mammalian selectable marker,wherein said marker cassette is flanked on each side by mammaliangene-specific sequences homologous for a portion of the gene to betargeted; d) transforming yeast cells with the second expressionconstruct and the DNA from the positive clone; e) selecting thetransformed yeast cells for expression of the first and second yeastselectable markers; and f) isolating the targeting vector produced bythe recombination between the shuttle vector and the second expressionconstruct.
 28. The method of claim 27, wherein said positive poolscomprising the target gene are selected by PCR analysis of the poolswith gene-specific PCR primers wherein amplification of the PCR productsis indicative of the pool comprising the target gene of interest. 29.The method of claim 27, wherein said gene-specific flanking sequenceseach comprises at least about 20 nucleotides.
 30. The method of claim27, wherein said gene specific flanking sequences each comprises fromabout 35 to about 400 nucleotides.
 31. The method of claim 27, whereinsaid fragment of genomic DNA comprises from about 0.5 kb to about 5 kbof DNA on each side of a site in said gene to be targeted.
 32. Themethod of claim 27, wherein said fragment of genomic DNA comprises atleast about 1 kb of genomic DNA on each side of a site in said gene tobe targeted.
 33. The method of claim 27, wherein said second yeastselectable marker is selected from the group consisting of TRP1, His,Ura3, Ade, Arg and Leu2.
 34. The method of claim 27, wherein said secondmammalian selectable marker is selected from the group consisting ofconsisting of thymidine kinase, neomycin resistance, hygromycinresistance, Salmonella HisD and puromycin-acetyl transferase.
 35. Themethod of claim 27, wherein said marker cassette comprises Ura3 as thesecond yeast selectable marker and the neomycin resistance gene as thesecond mammalian selectable marker.
 36. A method of preparing a genomiclibrary comprising: a) preparing size selected genomic DNA; b) preparinga shuttle vector comprising said genomic DNA and a yeast vector; c)amplifying said shuttle vector in transformed bacterial host cells; andd) arraying said transformed bacterial host cells into pools wherein thebacterial host cells comprise said shuttle vectors and wherein saidpools comprise a library of genomic fragments of pre-selected sizes. 37.A genomic library prepared according to the method of claim
 36. 38. Thegenomic library of claim 37, wherein said library is a mammalian genomiclibrary.
 39. The genomic library of claim 38, wherein said mammaliangenomic library is a mouse genomic library.